BTB and TAZ domain protein BT4 positively regulates the resistance to Botrytis cinerea in Arabidopsis

ABSTRACT BT4 gene was identified to play an important role in Arabidopsis resistance to pst DC3000 in preliminary studies. However, the specific function and molecular mechanism of BT4 gene in regulation of Arabidopsis resistance to Botrytis cinerea had not been described to date. In this study, we found that the expression of BT4 was induced by wounding and B. cinerea inoculation in Arabidopsis. After inoculated with B. cinerea, T-DNA insertion mutants of the BT4 gene, bt4, showed significant susceptibility symptoms, whereas no significant symptoms were found in wild-type (WT), the complemented transgenic plants (CE), and the overexpression transgenic plants (OE). After inoculated with B. cinerea, the expression levels of JAR1 and PDF1.2 genes in bt4 mutant were induced; however, the expression levels of these genes in bt4 mutant were significantly lower than those in the WT, CE, and OE. These results indicated that the BT4 positively regulate the expression of genes in JA/ET signaling pathways. Therefore, the BT4 may be involved in the regulation of JA/ET signaling pathways to affect Arabidopsis resistance to B. cinerea.


Introduction
Gray mold caused by Botrytis cinerea is a world-wide plant disease, which seriously affects the yield and quality of crops. The isolation of resistant-related genes against B. cinerea and the molecular mechanism of its diseaseresistant provide the theoretical basis for the guidance of disease control and research of the molecular mechanism of interaction between plants and pathogens. At present, a lot of resistance-related genes of Arabidopsis against B. cinerea were identified. These genes regulate the resistance of Arabidopsis to B. cinerea by regulating SA and JA/ET signaling pathways. ERF1 is the activation factor of JA/ET signaling pathway in Arabidopsis. Overexpression of ERF1 could activate the expression of resistance-related genes PDF1. 2 and ChiB and increase resistance to B. cinerea. 1 In Arabidopsis MYB30 is a positive regulator of hypersensitive response (HR) in Arabidopsis. Overexpression of MYB30 in tobacco and Arabidopsis were more resistant to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). 2 MYB46 could bound to the cis site of the promoter of cell wall peroxidase encoding gene Ep5C in Arabidopsis to regulate the synthesis of secondary cell wall in vascular bundles, which affected the resistance of Arabidopsis to B. cinerea. 3 The BOS1 (MYB108) gene is mainly involved in JA signaling pathway of Arabidopsis. Loss of BOS1 displayed more sensitive to B. cinerea and increased the resistance to Pst DC3000. 4 ERF19 as a novel player in the mitigation of PTI, and highlights a potential role for NINJA in fine-tuning ERF19-mediated regulation of Arabidopsis against B. cinerea. 5 WRKY33 is a key transcriptional regulator of hormonal and metabolic processes toward B. cinerea infection and is essential for resistance. 6,7 Approximately 80 BTB proteins were identified in Arabidopsis, and several reports showed that BTB protein play a variety of functions in organisms, including transcriptional activation and inhibition, 8 regulation of cytoskeleton, 9 regulation of ion channels, 10 and ubiquitination of protein. 11 Five BT proteins (BT1-BT5) with BTB and TAZ domains were grouped into a small subfamily that interacts with calmodulin. Among them, BT1, BT2, and BT4 were found to bind to bromo-domain transcriptional regulators and the BT2 promoter binds to transcription factor TELOMERASE ACTIVATOR1 (TAC1) to regulate telomerase activity in mature vegetative organs. 12,13 Some genes containing BTB domains were involved in plant disease resistance, such as NPR1 of Arabidopsis. 14 Previously, the BT4 gene, encoding a transcriptional regulator with BTB (broad-complex, tramtrack, and brica-brac) and TAZ (a transcriptional adapter zinc finger) domains, was isolated and identified as a resistant-related gene in response to Pst DC3000; meanwhile, BT4 also could be induced by SA and JA 15  investigated the function of BT4 in Arabidopsis resistance to B. cinerea and further analyzed the probable mechanism of BT4 gene regulation in Arabidopsis resistance to B. cinerea.

BT4 positively regulates the resistance to B. cinerea in Arabidopsis
Wounding provides nutrients to pathogens and facilitates their entry into the tissue and subsequent infection. Plants have evolved constitutive and induced defense mechanisms to properly respond to wounding and prevent infection. To explore the expression level of BT4 under wounding stress, we analyzed the datasets related to wounding stress from the GEO database (GSE101422). It was found that BT4 was significantly induced by wounding across different timepoint (Figure 1a).  (Figure 1b). Trypan blue and DAB staining revealed that there were numerous dead cells and ROS in the leaves of the bt4 mutant inoculated with B. cinerea, with fewer dead cells and ROS were observed in leaves of Col-0, CE and OE plants (Figure 1b). Consistent with this phenotype, the expression levels of BcACTIN were significantly upregulated in bt4 mutant (Figure 1c), and the content of chlorophyll were decreased obviously in bt4 mutant (Figure 1d). In addition, the plant hormone JA in bt4 mutants were also detected, and it was found that the contents of JA were also affected by the expression of BT4 gene. The contents of the plant hormone JA were significantly reduced in bt4, partially recovered in CE, and the highest in OE, exceeding Col-0 (Figure 2a). The results showed that bt4 mutant had strong susceptibility to B. cinerea, and indicated that the BT4 gene played a positive role in Arabidopsis resistance to B. cinerea. and LOX2 were obviously reduced by B. cinerea in the Col-0, bt4, CE, and OE plants (Figure 2(b-f)). However, the expression levels of these genes were significantly down-regulated in bt4 mutants compared to those in the Col-0, CE, and OE plants. These results suggested that the BT4 gene might affect the expression of JA/ET signaling pathway genes in response to B. cinerea infection.

Conclusion
The JA signaling pathways play critical roles in protecting plant against pathogens. 16 JA and ET are also involved in induced systemic resistance of plants. 17 In this study, we found that wounding could induce the expression level of BT4 gene. After inoculation with B. cinerea, the expression levels of key genes of the JA signaling pathways in the bt4 mutant were significantly lower than those in the wild-type, CE, and OE mutant. These results indicated that the BT4 gene might be involved in the regulation of JA signaling pathways and further regulated the resistance to B. cinerea in Arabidopsis.  leaves were stained with trypan blue and DAB, and used for and relative expression level of B. cinerea BcACTIN and determination of chlorophyll content.

RNA extraction and real-time PCR analysis of gene expression
Total RNA of Arabidopsis plants were extracted with Trizol regent (TaKaRa, Dalian, China) according to the manufacturer's instructions and treated with RNase-free DNase I (Omega, USA) to remove genomic DNAs. The first strand cDNA was obtained using PrimeScript RT regent kit (TaKaRa, Dalian, China). The Real-time PCR reaction was done on a CFX96 real-time PCR system (BioRad, Hercules, CA, USA) in 10 μL reactions containing 0.2 μL SYBR Premix Ex Taq TM (TaKaRa, Dalian, China) and each gene-specific primer (Table  S1). Relative gene expression levels were calculated using 2 −CT method with three independent biological replicates.

Disclosure statement
No potential conflict of interest was reported by the authors.